The ultimate goal of these studies is to develop effective strategies for improving the outcome of injuries to the spinal cord in humans. A moderate-severity contusion injury model in rats, characterize as are most injuries in humans by the interruption of axons that control lesion will not occur without treatment. This proposal first describes a series of three Aims comparing progressively more complex strategies to promote regeneration across a lesion site and into distal spinal cord. The strategy that maximizes regeneration will then be tested at delayed time points. A neuroprotective agent will be injected into all rats immediately following injury. In Aim 1, the lesion site will be modified at 1 week after injury by the transplantation, in medium containing fibrinogen and fibroblast growth factor-I, or Schwann cells or unsheathing glia (EG), or a 1:1 mixture of these regeneration-promoting cell types. A double-labeling retrograde tracing technique will be used to detect neurons in locomotion centers in the brain and brainstem which regenerate their axons into the lesion. At 12 weeks after injury the transplant type resulting in the regeneration of the highest number of axons into the lesion will be determined by counting the appropriately labeled neurons. In Aim 2, the most effective transplant from Aim 1 will be tested in the presence of increased levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the lesion. Axonal regeneration induced by 1) transplanted cells infected with adeno-associated viral (AAV) constructs coding for BDNF and NT-3, 2) uninfected cells transplanted with BDNF/NT-3 laden biodegradable microspheres, or 3) embryonic stem cells (naive or astrocyte-restricted) infected with a retroviral construct coding for a bi-functional neurotrophin with BDNF and NT-3 activity will be compared. In Aim 3, the most effective of these strategies will be combined with treatments to induce growth of the axons out of the lesion and into the distal cord. EG will be transplanted just distal to the lesion, alone or with the more effective of AAV-BDNF/AAV-NT-3 or BDNF/NT-3 laden microspheres injected mm distal to the lesion. The treatment resulting in the most axonal regeneration into the distal cord will be determined. In all studies, regeneration will also be assessed using anterograde tracing of axons from neurons in the hindlimb region of the cortex, in the lateral vestibular nucleus, and the reticular formation. Immunocytochemical methods will be used to classify the regenerating axons and to determine the expression of trkB and trkC receptors on responsive axons. Basso-Beattie-Bresnahan open field locomotion tests and electrophysiological analysis will also be performed. Importantly, in the fourth and final Aim, the response of severed locomotion-related axons to the most effective strategy defined in Aim 3 will be determined when cellular transplantation and neurotrophin treatments are delayed until 4 and 4 weeks after injury. An improved understanding of the change in potential of axons to regenerate with time after injury is critical for predicting the clinical merit of any regeneration-promoting strategy.